Qubit 2.0 fluorometer with qubit microrna assay kit

The RNA quality and quantity were verified according to MIQE guidelines^{20 (link)}. The RNA concentration was quantified by Qubit^® 2.0 Fluorometer with Qubit^® microRNA Assay Kit (Invitrogen, Cat. No. Q32880). An NGS was performed on the plasma of 8 patients at three time points. Small RNA transcripts were converted into barcoded cDNA libraries. Library preparation was performed as previously reported^{21 (link)} using the NEBNext Multiplex small RNA Library Prep Set (Cat No NEB#E7560) for Illumina and run on the NextSeq500 (Illumina Inc., USA).

Total RNA was extracted using miRNeasy Serum/Plasma Kit (Qiagen, Cat. No 217184) following the manufacturer's instruction. The RNA quality and quantity were verified according to MIQE guidelines (27 (link)). For all samples, RNA concentration was quantified by Qubit^® 2.0 Fluorometer with ^Qubit® microRNA Assay Kit (Invitrogen, Cat. No. Q32880).
A pilot sequencing was performed on 3 healthy (Supplementary Table 1, no. 15 right, 19 right, and 20 right) and 3 otitis-affected samples (Supplementary Table 1, no. 32 left, 35 left, and 41 right). Small RNA transcripts were converted into barcoded cDNA libraries. Library preparation was performed as previously reported (28 (link)) using the NEBNext Multiplex small RNA Library Prep Set (Cat. No. NEB#E7560) for Illumina and run on the NextSeq500 (Illumina Inc., USA).

Total RNA from plasma exosomes was extracted with the miRNeasy plasma/serum mini kit (Qiagen) using the QiaCube extractor (Qiagen). RNA from stool was extracted using the Stool Total RNA Purification Kit (Norgen Biotek Corp). Total RNA from urine was extracted with Urine microRNA Purification kit (Norgen biotek corp), following the manufacturer’s standard protocol.
RNA from cervical scrape was extracted from samples stored in STM or RNA-later, using the miRCURY™ RNA Isolation Kit - Cell & Plant (Exiqon) following manufacturer`s protocol.
RNA quality and quantity was verified according to MIQE guidelines (http://miqe.gene-quantification.info/). For all samples, RNA concentration was quantified by Qubit^® 2.0 Fluorometer with Qubit^® microRNA Assay Kit (Invitrogen).